One of the major problems of gene therapy is the effective delivery of the therapeutic agent into target cells in vitro or in vivo. Although viral vectors have certain advantages, including high levels of transfection, or efficient and stable integration of foreign DNA into a wide range of host genomes, they suffer from several problems including immunogenicity, toxicity, difficulty of large-scale production, size limit of the exogenous DNA, random integration into the host genome, and the risks of inducing tumorigenic mutations and/or generating active viral particles through recombination (Singhal, A. and Huang, L., (1994) In: Wolf, J. A.(ed), Gene Therapeutics: Methods and Applications of Direct Gene Transfer. Birkhauser: Boston, pp118-142: Lee, R. J., and L. Huang, (1996) J. Biol. Chem. 271:8481-8487). These limitations of viral vectors have prompted investigators to try to improve methods of non-viral gene delivery. (Treco, D. A. and R. F. Selden, (1995) Mol. Med. Today 1:314-321).
Cationic liposomes have been used extensively for in vitro and in vivo gene delivery, and constitute a viable alternative to viral gene delivery vehicles. (Singhal A., and L. Huang, supra; Hug P and R. G. Sleight, (1991) Biochim Biophys Acta 1097: 1-17: Lasic D. D. and N. S. Templeton, (1996) Adv. Drug Deliv. Rev. 20: 221-266). Using this delivery system, relatively stable expression has been achieved in a number of tissues. (Liu, Y., et al., (1995) J Biol Chem 270: 24864-24870: Thierry, A. R., et al., (1995) Proc Natl Acad Sci USA 92: 9742-9746: Takehara, T., et al., (1995) Hepatology 21:46-751.)
The efficiency of cationic liposome transfection was improved through the use of two different approaches. The first approach was based on the promotion of cellular internalization of the cationic liposome-DNA complexes through receptor-mediated endocytosis. For this purpose iron-saturated human transferrin, and other receptor-mediated ligands, were associated with the lipid-DNA complexes, referred to herein as "lipoplexes" (Felgner,et al., (1997) Hum. Gene Ther. 8: 511-512) at different (+/-) charge ratios. Transferrin is a useful ligand that binds to a cell-surface receptor expressed by most proliferating cells, with particularly high expression on erythroblasts and tumor cells (Wagner et al., (1994) Adv. Drug. Del. Rev. 14:113-136). Another report indicated that associating transferrin with cationic liposomes also enhanced transfection of HeLa cells (Cheng, P. W. (1996) Hum. Gene Ther. 7:275-282). The use of transferrin as a receptor-ligand in lipoplex-mediated transfections was also disclosed by Cheng, P. W. in PCT publication WO 97/28817, incorporated herein by reference is referred to hereinafter as "Cheng (1997)".
The second approach was based on the association of endosome disrupting agents to the lipoplexes with the purpose of facilitating the cytoplasmic release of DNA from endosomes, thus preventing its lysosomal degradation and therefore enhancing transfection. Two different synthetic fusogenic peptides, "GALA" and the influenza virus hemagglutinin HA2 N-terminal peptide (hereinafter, "HA-2"), both low pH-activated rrtmbrane-active peptides, were used for that purpose (Simoes, S. et al.(1998) Gene Ther., in press).
The use of receptor-specific ligands as a targeting protein in liposomal delivery vehicles in vitro and in vivo presents several problems. Receptor-specific ligands are relatively rare molecules and incur considerable expense in isolating and collecting an adequate supply. Receptor-ligands are invariably potent effectors of biological response. Use of such molecules in lipoplexes increase their concentration in the cellular milieu and therefore pose a potentially serious threat of adverse or unwanted side-effects. Finally, studies have shown that high concentrations of blood serum can inhibit the efficiency of ligand-mediated lipoplexes, thereby raising serious questions as to their utility in an in vivo environment. (Cheng (1997).